Resistance to a novel antichlamydial compound is mediated through mutations in Chlamydia trachomatis secY.

Abstract

A novel and quantitative high-throughput screening approach was explored as a tool for the identification of novel compounds that inhibit chlamydial growth in mammalian cells. The assay is based on accumulation of a fluorescent marker by intracellular chlamydiae. Its utility was demonstrated by screening 42,000 chemically defined compounds against Chlamydia caviae GPIC. This analysis led to the identification of 40 primary-hit compounds. Five of these compounds were nontoxic to host cells and had similar activities against both C. caviae GPIC and Chlamydia trachomatis. The inhibitory activity of one of the compounds, (3-methoxyphenyl)-(4,4,7-trimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-C]quinolin-1-ylidene)amine (MDQA), was chlamydia specific and was selected for further study. Selection for resistance to MDQA led to the generation of three independent resistant clones of C. trachomatis. Amino acid changes in SecY, a protein involved in Sec-dependent secretion in Gram-negative bacteria, were associated with the resistance phenotype. The amino acids changed in each of the resistant mutants are located in the predicted central channel of a SecY crystal structure, based on the known structure of Thermus thermophilus SecY. These experiments model a process that can be used for the discovery of antichlamydial, anti-intracellular, or antibacterial compounds and has led to the identification of compounds that may have utility in both antibiotic discovery and furthering our understanding of chlamydial biology.

Concentration-dependent inhibition of chlamydial infections by different compounds in the SIGA library. C. caviae GPIC-infected cells were treated with half-log dilutions of each of 5 compounds identified as chlamydial inhibitors in the high-throughput screen and quantitated at 24 h postinfection (hpi) using C6-NBD-ceramide. Compound 4 is included as an example of a compound with no effect in the assay. Fluorescence readings (y axis) are plotted against each compound at different concentrations (x axis).

Immunofluorescence images of MDQA-treated C. caviae GPIC-, C. psittaci CP3-, and C. trachomatis L2-434-infected cells. McCoy cells were infected with each of the following strains and grown in the presence of either 1% DMSO as a vehicle control (A, C, E) or with MDQA at 10 μM (B, D, F) before fixation at each indicated time point. Panels A and B are C. psittaci CP3-infected cells fixed at 72 h. Panels C and D are C. trachomatis L2-434-infected cells fixed at 42 h. Panels E and F are C. caviae-infected cells fixed at 42 h. Scale bar = 10 μm.

qPCR of sensitive and resistant C. trachomatis L2-434 strains grown in the presence of MDQA. C. trachomatis L2-434-sensitive and C. trachomatis L2-434-resistant (clone 1) strains were cultured in McCoy cell monolayers in the presence or absence of MDQA at various concentrations (x axis). Infections were lysed at 40 hpi, and genome copies/ml were determined by qPCR (y axis).

Amino acid changes in SecY that are associated with MDQA resistance in C. trachomatis L2-434. Resistant strains were maintained in culture until the concentration exceeded the sensitive strain MIC by at least 2-fold before whole-genome sequencing or gene-specific (secY) sequencing. Three different mutations leading to amino acid substitutions in SecY were identified in 3 independently generated resistant mutants.

Predicted 3D structure of the chlamydial SecY translocon. The amino acid sequence of C. trachomatis L2-434 SecY was submitted to I-TASSER threading software to obtain a predicted 3D image. A top and side view of the predicted C. trachomatis SecY structure (A) is shown next to the crystallized SecY protein from T. thermophilus (B). The mutated amino acids of the MDQA-resistant C. trachomatis strains (plus each flanking residue) are highlighted on each structure, as well as those residues homologous to the C. trachomatis mutations on the crystallized T. thermophilus protein. Amino acid substitutions are highlighted as follows: alanine-to-serine substitution at amino acid 45 (red), alanine-to-proline substitution at amino acid 246 (blue), and alanine-to-proline substitution at amino acid 420 (orange).